Projects 1 and 2 of this program project will use expression library immunization (ELI) and DNA microarraying respectively to identify T. cruzi genes which are capable of eliciting protection from lethal infection when delivered as DNA in a mammalian expression plasmid. Project 3 will more fully access the vaccine potential of combinations of genes delivered with and without genetic cytokine adjuvants. The goal of this project is to determine if any of the vaccine candidates identified and characterized in Projects 1-3 are the target of immune responses in humans and if these responses correlated with clinical status of the infected individuals. To allow for the efficient and economic analysis of immune responses of human patients to multiple genes and gene products, we will develop systems for protein expression and targeting, and immunoassays for quantitative determination of antibody, CD4+ and CD8+ T cell responses. Inserts from the genomic library used in projects 1 and 2 will be recloned into bacterial expression vectors with and without membrane translocating sequences (MTS) to facilitate antigen presentation via class I MHC. ELISA and ELISPOT assay systems will be refined and optimized for measurement responses of a large group of donors to at least 20 candidate vaccine molecules. Quantitative humoral and cell mediated immune responses, including titers for antigen-specific immunoglobulin levels and frequencies of antigen-specific CD4+ and CD8+ cytokine-producing cells will be obtained. The presence and potency of these antigen specific responses will be correlated with the disease status of a selected group of human patients. The ultimate goal will be to determine if the quality or quantity of the immune response to any particular candidate vaccine molecules correlates with the disease status of the patients. These experiments will begin to allow us to address the concern that heightened immune responses to particular parasite antigens (as might occur naturally or induced through vaccination) will exacerbate rather than ameliorate disease.